Ubiquitin-dependent proteolysis plays a fundamental role in cell cycle regulation. Destruction of cyclin is required for cells to complete mitosis and evidence has recently been presented implicating the ubiquitin (Ub) pathway in this event. Four years ago this lab identified and purified a 26S protease capable of degrading ubiquitin-lysozyme conjugates. We proposed that the 26S protease was assembled from a well-known 20S protease (the multicatalytic protease or MCP) and polypeptides that confer ATP-dependence and ubiquitin recognition to the final complex. This application deals with the further characterization of this large ATP- stimulated multisubunit degradative enzyme. The isolation and sequencing of cDNAs that encode polypeptides in the 26S proteolytic particle will be continued. To study the assembly and disassembly of the 26S complex we have prepared fully active, fluoresceinated (F) 20S proteases and have shown that they can readily assemble into active 26S particles. We will continue to examine assembly/disassembly with the specific intention of testing "ribosome" versus "garbage disposal" models for protease mechanism. F-labeled 20S protease will also be employed to determine whether its subunits are in a state of dynamic equilibrium both in vitro and in living culture cells. Using photo-crosslinking approaches we identified two 26S subunits that bind ATP. Incubation of the 26S protease with gamma-32PO4- ATP produced three phosphorylated subunits. One of these is a 32K subunit in the 20S protease; we have evidence that another is a novel protein kinase inherent to the 26S particle. The role of phosphorylation in protease activity will be studied further. We will also use photo- crosslinking to identify 26S components that bind ubiquitin. Diubiquitin molecules linked by an isopeptide bond at lysine 48 will be derivatized and photocrosslinked to the 26S complex. Two years ago we developed a fast and very cheap method for synthesizing peptides as ubiquitin carboxyl extensions. This technology will be used to prepare chain-specific antibodies to components in the 26S complex and to prepare tethered substrates for probing active sites in the 20S protease. Finally, two separate enzymes that accurately process pentaUb to Ub have been identified. We will purify each enzyme, and we will continue our studies on poly-ubiquitin metabolism.